K. Theissinger, Anna Kästel, Vasco Elbrecht, Jenny Makkonen, S. Michiels, S. Schmidt, Stefanie Allgeier, F. Leese, C. Brühl
Chironomids have been proposed as important indicators for monitoring freshwater ecosystems, however, morphological determination is very challenging. In this study, we investigated the effectiveness of metabarcoding for chironomid diversity assessment and tested the retrieved chironomid operational taxonomic units (OTUs) for possible changes in relative abundance and species diversity in relation to mosquito control actions in four temporary wetlands. The biocide Bacillus thuringiensis var. israelensis (Bti) is widely applied for mosquito control in temporary wetlands of the German Upper Rhine Valley. Even though Bti is considered environmentally friendly, several studies have shown non-target effects on chironomids, a key food resource in wetland ecosystems Three of the studied wetlands were, for the first year after 20 years of Bti treatment, partly left Bti-untreated in a split field design, and one wetland has never been treated with Bti. Our metabarcoding approach detected 54 chironomid OTUs across all study sites, of which 70% could be identified to species level comparisons against the BOLD database. We showed that metabarcoding increased chironomid species determination by 70%. However, we found only minor significant effects of Bti on the chironomid community composition. Subsequent studies will have to address if and how the chironomid community composition might change in the now Bti-untreated temporary wetlands to assess effects of Bti.
摇尾虫已被提出作为监测淡水生态系统的重要指标，但其形态测定非常具有挑战性。在本研究中，我们研究了元条形码技术对手摇蝇多样性评价的有效性，并测试了在4个临时湿地中检索到的手摇蝇操作分类单位(OTUs)相对丰度和物种多样性可能与蚊虫控制行动有关的变化。杀菌剂苏云金芽孢杆菌(Bacillus thuringiensis var. israelensis, Bti)被广泛应用于德国上莱茵河谷临时湿地的蚊虫防治。尽管Bti被认为是环境友好型的，但一些研究表明，Bti对湿地生态系统中重要的食物资源——手拟虫有非靶效应。在研究的三个湿地中，在Bti处理20年后的第一年，部分湿地在分田设计中未处理Bti，还有一个湿地从未处理过Bti。我们的元条形码方法在所有研究地点检测到54个chironomid OTUs，其中70%可以与BOLD数据库进行物种水平的比较。我们发现元条形码使手拟虫的种类确定率提高了70%。然而，我们发现Bti对摇蚊群落组成的影响很小。后续的研究将必须解决在目前未经Bti处理的临时湿地中，摇尾虫群落组成是否以及如何发生变化，以评估Bti的影响。
{"title":"Corrigendum: Using DNA metabarcoding for assessing chironomid diversity and community change in mosquito controlled temporary wetlands. MBMG 2: e21060. https://doi.org/10.3897/mbmg.2.21060","authors":"K. Theissinger, Anna Kästel, Vasco Elbrecht, Jenny Makkonen, S. Michiels, S. Schmidt, Stefanie Allgeier, F. Leese, C. Brühl","doi":"10.3897/mbmg.4.60854","DOIUrl":"https://doi.org/10.3897/mbmg.4.60854","url":null,"abstract":"Chironomids have been proposed as important indicators for monitoring freshwater ecosystems, however, morphological determination is very challenging. In this study, we investigated the effectiveness of metabarcoding for chironomid diversity assessment and tested the retrieved chironomid operational taxonomic units (OTUs) for possible changes in relative abundance and species diversity in relation to mosquito control actions in four temporary wetlands. The biocide Bacillus thuringiensis var. israelensis (Bti) is widely applied for mosquito control in temporary wetlands of the German Upper Rhine Valley. Even though Bti is considered environmentally friendly, several studies have shown non-target effects on chironomids, a key food resource in wetland ecosystems Three of the studied wetlands were, for the first year after 20 years of Bti treatment, partly left Bti-untreated in a split field design, and one wetland has never been treated with Bti. Our metabarcoding approach detected 54 chironomid OTUs across all study sites, of which 70% could be identified to species level comparisons against the BOLD database. We showed that metabarcoding increased chironomid species determination by 70%. However, we found only minor significant effects of Bti on the chironomid community composition. Subsequent studies will have to address if and how the chironomid community composition might change in the now Bti-untreated temporary wetlands to assess effects of Bti.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46669511","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Characterization of biodiversity from environmental DNA samples and bulk metabarcoding data is hampered by off-target sequences that can confound conclusions about a taxonomic group of interest. Existing methods for isolation of target sequences rely on alignment to existing reference barcodes, but this can bias results against novel genetic variants. Effectively parsing targeted DNA barcode data from off-target noise improves the quality of biodiversity estimates and biological conclusions by limiting subsequent analyses to a relevant subset of available data. Here, we present Alfie, a Python package for the alignment-free classification of cytochrome c oxidase subunit I (COI) DNA barcode sequences to taxonomic kingdoms. The package determines k-mer frequencies of DNA sequences, and the frequencies serve as input for a neural network classifier that was trained and tested using ~58,000 publicly available COI sequences. The classifier was designed and optimized through a series of tests that allowed for the optimal set of DNA k-mer features and optimal machine learning algorithm to be selected. The neural network classifier rapidly assigns COI sequences of varying lengths to kingdoms with greater than 99% accuracy and is shown to generalize effectively and make accurate predictions about data from previously unseen taxonomic classes. The package contains an application programming interface that allows the Alfie package’s functionality to be extended to different DNA sequence classification tasks to suit a user’s need, including classification of different genes and barcodes, and classification to different taxonomic levels. Alfie is free and publicly available through GitHub (https://github.com/CNuge/alfie) and the Python package index (https://pypi.org/project/alfie/).
{"title":"Alignment-free classification of COI DNA barcode data with the Python package Alfie","authors":"C. Nugent, S. Adamowicz","doi":"10.3897/mbmg.4.55815","DOIUrl":"https://doi.org/10.3897/mbmg.4.55815","url":null,"abstract":"Characterization of biodiversity from environmental DNA samples and bulk metabarcoding data is hampered by off-target sequences that can confound conclusions about a taxonomic group of interest. Existing methods for isolation of target sequences rely on alignment to existing reference barcodes, but this can bias results against novel genetic variants. Effectively parsing targeted DNA barcode data from off-target noise improves the quality of biodiversity estimates and biological conclusions by limiting subsequent analyses to a relevant subset of available data. Here, we present Alfie, a Python package for the alignment-free classification of cytochrome c oxidase subunit I (COI) DNA barcode sequences to taxonomic kingdoms. The package determines k-mer frequencies of DNA sequences, and the frequencies serve as input for a neural network classifier that was trained and tested using ~58,000 publicly available COI sequences. The classifier was designed and optimized through a series of tests that allowed for the optimal set of DNA k-mer features and optimal machine learning algorithm to be selected. The neural network classifier rapidly assigns COI sequences of varying lengths to kingdoms with greater than 99% accuracy and is shown to generalize effectively and make accurate predictions about data from previously unseen taxonomic classes. The package contains an application programming interface that allows the Alfie package’s functionality to be extended to different DNA sequence classification tasks to suit a user’s need, including classification of different genes and barcodes, and classification to different taxonomic levels. Alfie is free and publicly available through GitHub (https://github.com/CNuge/alfie) and the Python package index (https://pypi.org/project/alfie/).","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":"24 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91395760","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Shunsuke Matsuoka, Yoriko Sugiyama, Hirotoshi Sato, Izumi Katano, Ken Harada, H. Doi
Growing evidence has revealed high diversity and spatial heterogeneity of fungal communities in local habitats of terrestrial ecosystems. Recently, the analysis of environmental DNA has been undertaken to study the biodiversity of organisms, such as animals and plants, in both aquatic and terrestrial habitats. In the present study, we investigated fungal DNA assemblages and their spatial structure using environmental DNA metabarcoding targeting the internal transcribed spacer 1 (ITS1) region of the rRNA gene cluster in habitats across different branches of rivers in forest landscapes. A total of 1,956 operational taxonomic units (OTUs) were detected. Of these, 770 were assigned as Ascomycota, 177 as Basidiomycota, and 38 as Chytridiomycota. The river water was found to contain functionally diverse OTUs of both aquatic and terrestrial fungi, such as plant decomposers and mycorrhizal fungi. These fungal DNA assemblages were more similar within, rather than between, river branches. In addition, the assemblages were more similar between spatially closer branches. This spatial structuring was significantly associated with geographic distances but not with vegetation of the catchment area and the elevation at the sampling points. Our results imply that information on the terrestrial and aquatic fungal compositions of watersheds, and therefore their spatial structure, can be obtained by investigating the fungal DNA assemblages in river water.
{"title":"Spatial structure of fungal DNA assemblages revealed with eDNA metabarcoding in a forest river network in western Japan","authors":"Shunsuke Matsuoka, Yoriko Sugiyama, Hirotoshi Sato, Izumi Katano, Ken Harada, H. Doi","doi":"10.3897/mbmg.3.36335","DOIUrl":"https://doi.org/10.3897/mbmg.3.36335","url":null,"abstract":"Growing evidence has revealed high diversity and spatial heterogeneity of fungal communities in local habitats of terrestrial ecosystems. Recently, the analysis of environmental DNA has been undertaken to study the biodiversity of organisms, such as animals and plants, in both aquatic and terrestrial habitats. In the present study, we investigated fungal DNA assemblages and their spatial structure using environmental DNA metabarcoding targeting the internal transcribed spacer 1 (ITS1) region of the rRNA gene cluster in habitats across different branches of rivers in forest landscapes. A total of 1,956 operational taxonomic units (OTUs) were detected. Of these, 770 were assigned as Ascomycota, 177 as Basidiomycota, and 38 as Chytridiomycota. The river water was found to contain functionally diverse OTUs of both aquatic and terrestrial fungi, such as plant decomposers and mycorrhizal fungi. These fungal DNA assemblages were more similar within, rather than between, river branches. In addition, the assemblages were more similar between spatially closer branches. This spatial structuring was significantly associated with geographic distances but not with vegetation of the catchment area and the elevation at the sampling points. Our results imply that information on the terrestrial and aquatic fungal compositions of watersheds, and therefore their spatial structure, can be obtained by investigating the fungal DNA assemblages in river water.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41462589","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Rosetta C. Blackman, E. Mächler, F. Altermatt, A. Arnold, P. Beja, P. Boets, Bastian Egeter, Vasco Elbrecht, A. Filipe, J. Jones, Jan-Niklas Macher, M. Majaneva, F. Martins, Cesc Múrria, Kristian Meissner, J. Pawłowski, Paul Schmidt Yáñez, V. Zizka, F. Leese, B. Price, Kristy Deiner
Over the last decade, steady advancements have been made in the use of DNA-based methods for detection of species in a wide range of ecosystems. This progress has culminated in molecular monitoring methods being employed for the detection of several species for enforceable management purposes of endangered, invasive, and illegally harvested species worldwide. However, the routine application of DNA-based methods to monitor whole communities (typically a metabarcoding approach) in order to assess the status of ecosystems continues to be limited. In aquatic ecosystems, the limited use is particularly true for macroinvertebrate communities. As part of the DNAqua-Net consortium, a structured discussion was initiated with the aim to identify potential molecular methods for freshwater macroinvertebrate community assessment and identify important knowledge gaps for their routine application. We focus on three complementary DNA sources that can be metabarcoded: 1) DNA from homogenised samples (bulk DNA), 2) DNA extracted from sample preservative (fixative DNA), and 3) environmental DNA (eDNA) from water or sediment. We provide a brief overview of metabarcoding macroinvertebrate communities from each DNA source and identify challenges for their application to routine monitoring. To advance the utilisation of DNA-based monitoring for macroinvertebrates, we propose an experimental design template for a series of methodological calibration tests. The template compares sources of DNA with the goal of identifying the effects of molecular processing steps on precision and accuracy. Furthermore, the same samples will be morphologically analysed, which will enable the benchmarking of molecular to traditional processing approaches. In doing so we hope to highlight pathways for the development of DNA-based methods for the monitoring of freshwater macroinvertebrates.
{"title":"Advancing the use of molecular methods for routine freshwater macroinvertebrate biomonitoring – the need for calibration experiments","authors":"Rosetta C. Blackman, E. Mächler, F. Altermatt, A. Arnold, P. Beja, P. Boets, Bastian Egeter, Vasco Elbrecht, A. Filipe, J. Jones, Jan-Niklas Macher, M. Majaneva, F. Martins, Cesc Múrria, Kristian Meissner, J. Pawłowski, Paul Schmidt Yáñez, V. Zizka, F. Leese, B. Price, Kristy Deiner","doi":"10.3897/MBMG.3.34735","DOIUrl":"https://doi.org/10.3897/MBMG.3.34735","url":null,"abstract":"Over the last decade, steady advancements have been made in the use of DNA-based methods for detection of species in a wide range of ecosystems. This progress has culminated in molecular monitoring methods being employed for the detection of several species for enforceable management purposes of endangered, invasive, and illegally harvested species worldwide. However, the routine application of DNA-based methods to monitor whole communities (typically a metabarcoding approach) in order to assess the status of ecosystems continues to be limited. In aquatic ecosystems, the limited use is particularly true for macroinvertebrate communities. As part of the DNAqua-Net consortium, a structured discussion was initiated with the aim to identify potential molecular methods for freshwater macroinvertebrate community assessment and identify important knowledge gaps for their routine application. We focus on three complementary DNA sources that can be metabarcoded: 1) DNA from homogenised samples (bulk DNA), 2) DNA extracted from sample preservative (fixative DNA), and 3) environmental DNA (eDNA) from water or sediment. We provide a brief overview of metabarcoding macroinvertebrate communities from each DNA source and identify challenges for their application to routine monitoring. To advance the utilisation of DNA-based monitoring for macroinvertebrates, we propose an experimental design template for a series of methodological calibration tests. The template compares sources of DNA with the goal of identifying the effects of molecular processing steps on precision and accuracy. Furthermore, the same samples will be morphologically analysed, which will enable the benchmarking of molecular to traditional processing approaches. In doing so we hope to highlight pathways for the development of DNA-based methods for the monitoring of freshwater macroinvertebrates.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48151912","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Bonnie Bailet, A. Bouchez, A. Franc, J. Frigerio, F. Keck, S. Karjalainen, F. Rimet, S. Schneider, M. Kahlert
Diatoms are known to be efficient bioindicators for water quality assessment because of their rapid response to environmental pressures and their omnipresence in water bodies. The identification of benthic diatoms communities in the biofilm, coupled with quality indices such as the Indice de polluosensibilité spécifique (IPS) can be used for biomonitoring purposes in freshwater. However, the morphological identification and counting of diatoms species under the microscope is time-consuming and requires extensive expertise to deal with a constantly evolving taxonomy. In response, a molecular-based and potentially more cost-effective method has been developed, coupling high-throughput sequencing and DNA metabarcoding. The method has already been tested for water quality assessment with diatoms in Central Europe. In this study, we applied both the traditional and molecular methods on 180 biofilms samples from Northern Europe (rivers and lakes of Fennoscandia and Iceland). The DNA metabarcoding data were obtained on two different DNA markers, the 18S-V4 and rbcL barcodes, with the NucleoSpin Soil kit for DNA extraction and sequenced on an Ion Torrent PGM platform. We assessed the ability of the molecular method to produce species inventories, IPS scores and ecological status class comparable to the ones generated by the traditional morphology-based approach. The two methods generated correlated but significantly different IPS scores and ecological status assessment. The observed deviations are explained by presence/absence and abundance discrepancies in the species inventories, mainly due to the incompleteness of the barcodes reference databases, primer bias and strictness of the bioinformatic pipeline. Abundance discrepancies are less common than presence/absence discrepancies but have a greater effect on the ecological assessment. Missing species in the reference databases are mostly acidophilic benthic diatoms species, typical of the low pH waters of Northern Europe. The two different DNA markers also generated significantly different ecological status assessments. The use of the 18S-V4 marker generates more species inventories discrepancies, but achieves an ecological assessment more similar to the traditional morphology-based method. Further development of the metabarcoding method is needed for its use in environmental assessment. For its application in Northern Europe, completion and curation of reference databases are necessary, as well as evaluation of the currently available bioinformatics pipelines. New indices, fitted for environmental biomonitoring, should also be developed directly from molecular data.
{"title":"Molecular versus morphological data for benthic diatoms biomonitoring in Northern Europe freshwater and consequences for ecological status","authors":"Bonnie Bailet, A. Bouchez, A. Franc, J. Frigerio, F. Keck, S. Karjalainen, F. Rimet, S. Schneider, M. Kahlert","doi":"10.3897/MBMG.3.34002","DOIUrl":"https://doi.org/10.3897/MBMG.3.34002","url":null,"abstract":"Diatoms are known to be efficient bioindicators for water quality assessment because of their rapid response to environmental pressures and their omnipresence in water bodies. The identification of benthic diatoms communities in the biofilm, coupled with quality indices such as the Indice de polluosensibilité spécifique (IPS) can be used for biomonitoring purposes in freshwater. However, the morphological identification and counting of diatoms species under the microscope is time-consuming and requires extensive expertise to deal with a constantly evolving taxonomy. In response, a molecular-based and potentially more cost-effective method has been developed, coupling high-throughput sequencing and DNA metabarcoding. The method has already been tested for water quality assessment with diatoms in Central Europe. In this study, we applied both the traditional and molecular methods on 180 biofilms samples from Northern Europe (rivers and lakes of Fennoscandia and Iceland). The DNA metabarcoding data were obtained on two different DNA markers, the 18S-V4 and rbcL barcodes, with the NucleoSpin Soil kit for DNA extraction and sequenced on an Ion Torrent PGM platform. We assessed the ability of the molecular method to produce species inventories, IPS scores and ecological status class comparable to the ones generated by the traditional morphology-based approach. The two methods generated correlated but significantly different IPS scores and ecological status assessment. The observed deviations are explained by presence/absence and abundance discrepancies in the species inventories, mainly due to the incompleteness of the barcodes reference databases, primer bias and strictness of the bioinformatic pipeline. Abundance discrepancies are less common than presence/absence discrepancies but have a greater effect on the ecological assessment. Missing species in the reference databases are mostly acidophilic benthic diatoms species, typical of the low pH waters of Northern Europe. The two different DNA markers also generated significantly different ecological status assessments. The use of the 18S-V4 marker generates more species inventories discrepancies, but achieves an ecological assessment more similar to the traditional morphology-based method. Further development of the metabarcoding method is needed for its use in environmental assessment. For its application in Northern Europe, completion and curation of reference databases are necessary, as well as evaluation of the currently available bioinformatics pipelines. New indices, fitted for environmental biomonitoring, should also be developed directly from molecular data.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-06-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44082933","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
V. Valentin, Rimet Frédéric, D. Isabelle, Monnier Olivier, R. Yorick, Bouchez Agnès
Ecological status assessment of watercourses is based on the calculation of quality indices using pollution sensitivity of targeted biological groups, including diatoms. The determination and quantification of diatom species is generally based on microscopic morphological identification, which requires expertise and is time-consuming and costly. In Europe, this morphological approach is legally imposed by standards and regulatory decrees by the Water Framework Directive (WFD). Over the past decade, a DNA-based molecular biology approach has newly been developed to identify species based on genetic criteria rather than morphological ones (i.e. DNA metabarcoding). In combination with high throughput sequencing technologies, metabarcoding makes it possible both to identify all species present in an environmental sample and to process several hundred samples in parallel. This article presents the results of two recent studies carried out on the WFD networks of rivers of Mayotte (2013–2018) and metropolitan France (2016–2018). These studies aimed at testing the potential application of metabarcoding for biomonitoring in the context of the WFD. We discuss the various methodological developments and optimisations that have been made to make the taxonomic inventories of diatoms produced by metabarcoding more reliable, particularly in terms of species quantification. We present the results of the application of this DNA approach on more than 500 river sites, comparing them with those obtained using the standardised morphological method. Finally, we discuss the potential of metabarcoding for routine application, its limits of application and propose some recommendations for future implementation in WFD.
{"title":"Assessing pollution of aquatic environments with diatoms’ DNA metabarcoding: experience and developments from France water framework directive networks","authors":"V. Valentin, Rimet Frédéric, D. Isabelle, Monnier Olivier, R. Yorick, Bouchez Agnès","doi":"10.3897/mbmg.3.39646","DOIUrl":"https://doi.org/10.3897/mbmg.3.39646","url":null,"abstract":"Ecological status assessment of watercourses is based on the calculation of quality indices using pollution sensitivity of targeted biological groups, including diatoms. The determination and quantification of diatom species is generally based on microscopic morphological identification, which requires expertise and is time-consuming and costly. In Europe, this morphological approach is legally imposed by standards and regulatory decrees by the Water Framework Directive (WFD). Over the past decade, a DNA-based molecular biology approach has newly been developed to identify species based on genetic criteria rather than morphological ones (i.e. DNA metabarcoding). In combination with high throughput sequencing technologies, metabarcoding makes it possible both to identify all species present in an environmental sample and to process several hundred samples in parallel. This article presents the results of two recent studies carried out on the WFD networks of rivers of Mayotte (2013–2018) and metropolitan France (2016–2018). These studies aimed at testing the potential application of metabarcoding for biomonitoring in the context of the WFD. We discuss the various methodological developments and optimisations that have been made to make the taxonomic inventories of diatoms produced by metabarcoding more reliable, particularly in terms of species quantification. We present the results of the application of this DNA approach on more than 500 river sites, comparing them with those obtained using the standardised morphological method. Finally, we discuss the potential of metabarcoding for routine application, its limits of application and propose some recommendations for future implementation in WFD.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42129805","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Mohammed Ahmed, M. Back, T. Prior, G. Karssen, R. Lawson, Ian Adams, M. Sapp
For many organisms, there is agreement on the specific genomic region used for developing barcode markers. With nematodes, however, it has been found that the COI region designated for most animals lacks the taxonomic coverage (ability to amplify a diverse group of taxa) required of a metabarcoding marker. For that reason, studies on metabarcoding of nematodes thus far have utilized primarily regions within the highly conserved 18S ribosomal DNA. Two popular markers within this region are the ones flanked by the primer pairs NF1-18Sr2b and SSUF04-SSUR22. The NF1-18Sr2b primer pair, especially, has been critiqued as not being specific enough for nematodes leading to suggestions for other candidate markers while the SSUF04-SSUR22 region has hardly been tested on soil nematodes. The current study aimed to evaluate these two markers against other alternative ones within the 28S rDNA and the COI region for their suitability for nematode metabarcoding. The results showed that the NF1-18Sr2b marker could offer wide coverage and good resolution for characterizing soil nematodes. Sufficient availability of reference sequences for this region was found to be a significant factor that resulted in this marker outperforming the other markers, particularly the 18S-based SSUFO4-SSUR22 marker. None of the other tested regions compared with this marker in terms of the proportion of the taxa recovered. The COI-based marker had the lowest number of taxa recovered, and this was due to the poor performance of its primers and the insufficient number of reference sequences in public databases. In summary, this study highlights how dependent the success of metabarcoding is on the availability of a good reference sequence collection for the marker of choice as well as its taxonomic coverage.
{"title":"Metabarcoding of soil nematodes: the importance of taxonomic coverage and availability of reference sequences in choosing suitable marker(s)","authors":"Mohammed Ahmed, M. Back, T. Prior, G. Karssen, R. Lawson, Ian Adams, M. Sapp","doi":"10.3897/mbmg.3.36408","DOIUrl":"https://doi.org/10.3897/mbmg.3.36408","url":null,"abstract":"For many organisms, there is agreement on the specific genomic region used for developing barcode markers. With nematodes, however, it has been found that the COI region designated for most animals lacks the taxonomic coverage (ability to amplify a diverse group of taxa) required of a metabarcoding marker. For that reason, studies on metabarcoding of nematodes thus far have utilized primarily regions within the highly conserved 18S ribosomal DNA. Two popular markers within this region are the ones flanked by the primer pairs NF1-18Sr2b and SSUF04-SSUR22. The NF1-18Sr2b primer pair, especially, has been critiqued as not being specific enough for nematodes leading to suggestions for other candidate markers while the SSUF04-SSUR22 region has hardly been tested on soil nematodes. The current study aimed to evaluate these two markers against other alternative ones within the 28S rDNA and the COI region for their suitability for nematode metabarcoding. The results showed that the NF1-18Sr2b marker could offer wide coverage and good resolution for characterizing soil nematodes. Sufficient availability of reference sequences for this region was found to be a significant factor that resulted in this marker outperforming the other markers, particularly the 18S-based SSUFO4-SSUR22 marker. None of the other tested regions compared with this marker in terms of the proportion of the taxa recovered. The COI-based marker had the lowest number of taxa recovered, and this was due to the poor performance of its primers and the insufficient number of reference sequences in public databases. In summary, this study highlights how dependent the success of metabarcoding is on the availability of a good reference sequence collection for the marker of choice as well as its taxonomic coverage.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44376162","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
A. Nobile, D. Freitas‐Souza, Francisco J. Ruiz-Ruano, M. Nobile, G. O. D. Costa, F. P. Lima, J. Camacho, F. Foresti, Cláudio Oliveira
Knowledge of ichthyoplankton dynamics is extremely important for conservation management as it can provide information about preferential spawning sites, reproductive period, migratory routes and recruitment success, which can be used to guide management and conservation efforts. However, identification of the eggs and larvae of Neotropical freshwater fish is a difficult task. DNA barcodes have emerged as an alternative and highly accurate approach for species identification, but DNA barcoding can be time-consuming and costly. To solve this problem, we aimed to develop a simple protocol based on DNA metabarcoding, to investigate whether it is possible to detect and quantify all species present in a pool of organisms. To do this, 230 larvae were cut in half, one half was sequenced by the Sanger technique and the other half was used to compose six arrays with a pool of larvae that were sequenced using a next-generation technique (NGS). The results of the Sanger sequencing allowed the identification of almost all larvae at species level, and the results from NGS showed high accuracy in species detection, ranging from 83% to 100%, with an average of 95% in all samples. No false positives were detected. The frequency of organisms in the two methods was positively correlated (Pearson), with low variation among species. In conclusion, this protocol represents a considerable advance in ichthyoplankton studies, allowing a rapid, cost-effective, quali-quantitative approach that improves the accuracy of identification.
{"title":"DNA metabarcoding of Neotropical ichthyoplankton: Enabling high accuracy with lower cost","authors":"A. Nobile, D. Freitas‐Souza, Francisco J. Ruiz-Ruano, M. Nobile, G. O. D. Costa, F. P. Lima, J. Camacho, F. Foresti, Cláudio Oliveira","doi":"10.3897/mbmg.3.35060","DOIUrl":"https://doi.org/10.3897/mbmg.3.35060","url":null,"abstract":"Knowledge of ichthyoplankton dynamics is extremely important for conservation management as it can provide information about preferential spawning sites, reproductive period, migratory routes and recruitment success, which can be used to guide management and conservation efforts. However, identification of the eggs and larvae of Neotropical freshwater fish is a difficult task. DNA barcodes have emerged as an alternative and highly accurate approach for species identification, but DNA barcoding can be time-consuming and costly. To solve this problem, we aimed to develop a simple protocol based on DNA metabarcoding, to investigate whether it is possible to detect and quantify all species present in a pool of organisms. To do this, 230 larvae were cut in half, one half was sequenced by the Sanger technique and the other half was used to compose six arrays with a pool of larvae that were sequenced using a next-generation technique (NGS). The results of the Sanger sequencing allowed the identification of almost all larvae at species level, and the results from NGS showed high accuracy in species detection, ranging from 83% to 100%, with an average of 95% in all samples. No false positives were detected. The frequency of organisms in the two methods was positively correlated (Pearson), with low variation among species. In conclusion, this protocol represents a considerable advance in ichthyoplankton studies, allowing a rapid, cost-effective, quali-quantitative approach that improves the accuracy of identification.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43819240","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The Decapoda is one of the largest orders within the class Malacostraca, comprising approximately 14,000 extant species and including many commercially important species. For biodiversity monitoring in a non-invasive manner, a new set of PCR primers was developed for metabarcoding environmental DNA (eDNA) from decapod crustaceans. The new primers (herein named “MiDeca”) were designed for two conservative regions of the mitochondrial 16S rRNA gene, which amplify a short, hyper-variable region (153–184 bp, 164 bp on average) with sufficient interspecific variations. With the use of MiDeca primers and tissue-derived DNA extracts, we successfully determined those sequences (154–189 bp) from 250 species, placed in 186 genera and 65 families across the suborder Dendrobranchiata and 10 of the 11 infraorders of the suborder Pleocyemata. We also preliminarily attempted eDNA metabarcoding from natural seawater collected at Banda, Tateyama, the Pacific coast of central Japan and detected 42 decapod species including 34 and 8 species with sequence identities of > 98% and 80–98%, respectively. The results suggest the usefulness of eDNA metabarcoding with MiDeca primers for biodiversity monitoring of the decapod species. It appears, however, that further optimisation of primer sequences would still be necessary to avoid possible PCR dropouts from eDNA extracts.
{"title":"Development of a new set of PCR primers for eDNA metabarcoding decapod crustaceans","authors":"T. Komai, R. Gotoh, T. Sado, M. Miya","doi":"10.3897/MBMG.3.33835","DOIUrl":"https://doi.org/10.3897/MBMG.3.33835","url":null,"abstract":"The Decapoda is one of the largest orders within the class Malacostraca, comprising approximately 14,000 extant species and including many commercially important species. For biodiversity monitoring in a non-invasive manner, a new set of PCR primers was developed for metabarcoding environmental DNA (eDNA) from decapod crustaceans. The new primers (herein named “MiDeca”) were designed for two conservative regions of the mitochondrial 16S rRNA gene, which amplify a short, hyper-variable region (153–184 bp, 164 bp on average) with sufficient interspecific variations. With the use of MiDeca primers and tissue-derived DNA extracts, we successfully determined those sequences (154–189 bp) from 250 species, placed in 186 genera and 65 families across the suborder Dendrobranchiata and 10 of the 11 infraorders of the suborder Pleocyemata. We also preliminarily attempted eDNA metabarcoding from natural seawater collected at Banda, Tateyama, the Pacific coast of central Japan and detected 42 decapod species including 34 and 8 species with sequence identities of > 98% and 80–98%, respectively. The results suggest the usefulness of eDNA metabarcoding with MiDeca primers for biodiversity monitoring of the decapod species. It appears, however, that further optimisation of primer sequences would still be necessary to avoid possible PCR dropouts from eDNA extracts.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46443487","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Metagenomics has emerged as a central technique for studying the structure and function of microbial communities. Often the functional analysis is restricted to classification into broad functional categories. However, important phenotypic differences, such as resistance to antibiotics, are often the result of just one or a few point mutations in otherwise identical sequences. Bioinformatic methods for metagenomic analysis have generally been poor at accounting for this fact, resulting in a somewhat limited picture of important aspects of microbial communities. Here, we address this problem by providing a software tool called Mumame, which can distinguish between wildtype and mutated sequences in shotgun metagenomic data and quantify their relative abundances. We demonstrate the utility of the tool by quantifying antibiotic resistance mutations in several publicly available metagenomic data sets. We also identified that sequencing depth is a key factor to detect rare mutations. Therefore, much larger numbers of sequences may be required for reliable detection of mutations than for most other applications of shotgun metagenomics. Mumame is freely available online (http://microbiology.se/software/mumame).
{"title":"Mumame: a software tool for quantifying gene-specific point-mutations in shotgun metagenomic data","authors":"S. Magesh, Viktor Jonsson, J. Bengtsson-Palme","doi":"10.3897/mbmg.3.36236","DOIUrl":"https://doi.org/10.3897/mbmg.3.36236","url":null,"abstract":"Metagenomics has emerged as a central technique for studying the structure and function of microbial communities. Often the functional analysis is restricted to classification into broad functional categories. However, important phenotypic differences, such as resistance to antibiotics, are often the result of just one or a few point mutations in otherwise identical sequences. Bioinformatic methods for metagenomic analysis have generally been poor at accounting for this fact, resulting in a somewhat limited picture of important aspects of microbial communities. Here, we address this problem by providing a software tool called Mumame, which can distinguish between wildtype and mutated sequences in shotgun metagenomic data and quantify their relative abundances. We demonstrate the utility of the tool by quantifying antibiotic resistance mutations in several publicly available metagenomic data sets. We also identified that sequencing depth is a key factor to detect rare mutations. Therefore, much larger numbers of sequences may be required for reliable detection of mutations than for most other applications of shotgun metagenomics. Mumame is freely available online (http://microbiology.se/software/mumame).","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48525619","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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